Recombinant Bradyrhizobium japonicum Translation initiation factor IF-3 (infC)

What is Translation Initiation Factor IF-3 and what is its role in B. japonicum?

Translation Initiation Factor IF-3 is an essential bacterial protein that plays critical roles in protein synthesis. In B. japonicum, as in other bacteria, IF-3 consists of two domains (IF3C and IF3N) separated by a linker sequence. The protein performs several crucial functions in translation: it interferes with ribosomal subunit association, promotes codon-anticodon interaction in the P site, and ensures translation initiation fidelity . These functions are particularly important in nitrogen-fixing bacteria like B. japonicum, where precise protein synthesis is required for symbiotic interactions with host plants.

How does the structure of IF-3 relate to its function in B. japonicum?

The IF-3 protein in B. japonicum has a two-domain structure with N-terminal (IF3N) and C-terminal (IF3C) domains connected by a flexible linker. This structural arrangement is essential for its sequential binding to the ribosome. During ribosomal binding, the C-domain first contacts the platform region (near G700) of the 30S ribosomal subunit, followed by the N-domain binding to the P-decoding region (near A790) . This sequential binding mechanism allows IF-3 to effectively monitor the fidelity of translation initiation and prevent premature subunit association, which is crucial for accurate protein synthesis in this agriculturally important bacterium.

How is the infC gene organized in the B. japonicum genome?

The infC gene in B. japonicum is part of the core genome rather than being located on symbiosis islands or plasmids. The B. japonicum genome contains several genomic islands and two to three symbiosis islands (SI A, SI B, and SI C) depending on the strain, but translation-related genes like infC are typically part of the core genome maintenance functions . The genomic organization surrounding infC is highly conserved across B. japonicum strains, reflecting the essential nature of translation initiation factors for bacterial survival.

What expression systems are most effective for recombinant production of B. japonicum IF-3?

For recombinant production of B. japonicum IF-3, E. coli-based expression systems are typically most effective due to their well-established protocols and high yield potential. When designing expression constructs, researchers should consider codon optimization for E. coli, as B. japonicum has a relatively high GC content (63.34-63.55%) , which can result in rare codons that may impede efficient translation in E. coli. T7-based expression systems with temperature-inducible or IPTG-inducible promoters often provide good control over expression timing. Including affinity tags (His6, GST, or MBP) can facilitate purification while potentially enhancing solubility, particularly important for multi-domain proteins like IF-3.

What are the challenges in purifying functional recombinant B. japonicum IF-3?

Purifying functional recombinant B. japonicum IF-3 presents several challenges. The two-domain structure with a flexible linker can lead to proteolytic susceptibility during expression and purification . Researchers should incorporate protease inhibitors throughout the purification process and consider rapid purification protocols to minimize degradation. Since IF-3 interacts with RNA, contaminating nucleic acids can co-purify with the protein, necessitating high-salt washes or nuclease treatments. Additionally, maintaining the native conformation of both domains is crucial for functionality. Researchers should verify proper folding through circular dichroism spectroscopy and assess functionality through ribosome binding assays or translation initiation assays.

How can researchers verify the structural integrity of purified recombinant B. japonicum IF-3?

Verifying the structural integrity of purified recombinant B. japonicum IF-3 requires multiple complementary approaches. Circular dichroism (CD) spectroscopy can confirm secondary structural elements, while thermal shift assays can assess stability. Limited proteolysis followed by mass spectrometry can verify domain boundaries and linker integrity. Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) helps determine if the protein exists in the expected monomeric state. Ultimately, functional assays such as 30S ribosomal subunit binding (monitored by fluorescence anisotropy or surface plasmon resonance) provide the most relevant assessment of proper folding and activity . Researchers should also consider NMR spectroscopy to analyze the dynamic relationship between the N and C domains, as this is critical for the sequential binding mechanism of IF-3.

What methodologies are most effective for studying B. japonicum IF-3 interactions with the ribosome?

For studying B. japonicum IF-3 interactions with the ribosome, time-resolved chemical probing has proven highly effective. This technique allows researchers to follow the dynamic binding path of IF-3 on the 30S ribosomal subunit and its release upon 30S-50S association . Complementary approaches include cryo-electron microscopy to visualize IF-3-ribosome complexes, surface plasmon resonance or microscale thermophoresis to measure binding kinetics, and hydroxyl radical footprinting to map precise interaction sites. Fluorescence-based assays with labeled IF-3 variants can track conformational changes during binding. For in vivo studies, B. japonicum strains with modified infC genes can be analyzed for growth defects and translational fidelity, while ribosome profiling can reveal the impact of IF-3 variants on global translation patterns.

What strategies are effective for creating site-directed mutations in B. japonicum infC gene?

Creating site-directed mutations in the B. japonicum infC gene requires careful consideration of this bacterium's genetic characteristics. Due to B. japonicum's relatively low transformation efficiency, researchers should consider using suicide vectors that integrate into the genome via homologous recombination. For precise modifications, CRISPR-Cas9 systems adapted for Bradyrhizobium can be used, though these may require optimization of promoters and guide RNA design due to the high GC content (63.34-63.55%) of B. japonicum. When designing mutations, researchers should focus on conserved residues identified through comparative genomics of various Bradyrhizobium strains and species . As infC is an essential gene, conditional mutants or complementation strategies may be necessary to study mutations that potentially impair function. Verification of mutations should include both sequencing and functional assays to assess the impact on translation initiation.

How can researchers develop B. japonicum strains with modified IF-3 for studying translational regulation during symbiosis?

Developing B. japonicum strains with modified IF-3 for symbiosis studies requires strategies that maintain bacterial viability while allowing controlled alteration of translation initiation. Researchers should consider using inducible expression systems where the native infC gene is replaced with a modified version under control of a symbiosis-specific promoter. Alternative approaches include creating strains with dual infC genes (native and modified) where the modified version has an inducible dominant-negative effect. For studying IF-3 localization during symbiosis, fluorescent protein fusions can be valuable, though care must be taken to ensure the fusion does not impair function. When evaluating modified strains, researchers should comprehensively assess both free-living growth and symbiotic performance metrics, including nodule formation, nitrogen fixation rates, and plant growth parameters as observed with various B. japonicum strains in previous studies . Correlation of these phenotypes with translation efficiency measurements will provide insights into the role of IF-3 in symbiotic adaptation.

What considerations are important when comparing IF-3 function across different Bradyrhizobium species and strains?

When comparing IF-3 function across different Bradyrhizobium species and strains, researchers must consider several important factors. Phylogenetic relationships between strains significantly impact comparative analyses - for example, strains previously classified as B. japonicum group Ia have been reclassified as B. diazoefficiens, with USDA 110 as the type strain . Genomic context differences may affect infC expression or regulation, as genomic organization can vary between species despite core gene conservation . Researchers should employ multiple sequence alignment to identify conserved versus variable regions in IF-3 that might correlate with host specificity or symbiotic efficiency. Functional assays must be standardized across strains, controlling for growth conditions that might affect translation. When expressing recombinant IF-3 from different strains, codon optimization strategies may need adjustment based on GC content variations between strains. Finally, translation regulation should be examined in the context of each strain's symbiotic properties, as observed in studies comparing strains like FA3, USDA110, and others with varying effects on plant growth and nitrogen fixation .

How might structural differences in IF-3 between B. japonicum and other bacteria be exploited for antimicrobial development?

While IF-3 is highly conserved due to its essential function, subtle structural differences between B. japonicum IF-3 and that of pathogenic bacteria could be exploited for selective antimicrobial development. Researchers should perform detailed structural comparisons focusing on the binding interfaces between IF-3 domains and ribosomal components, particularly the contacts near G700 and A790 . Molecular dynamics simulations can identify binding pocket differences that might accommodate selective inhibitors. Since B. japonicum is beneficial for agriculture, compounds that selectively inhibit pathogen IF-3 while sparing B. japonicum IF-3 could represent novel antimicrobials compatible with agricultural applications. Researchers should establish high-throughput screening assays based on IF-3 binding to ribosomes from different species to identify selective inhibitors. Structure-based drug design approaches targeting the unique features of pathogen IF-3 could lead to compounds that preserve beneficial soil microbiota while targeting pathogens.

What is the potential impact of IF-3 variants on translation efficiency during environmental stress in B. japonicum?

Environmental stress significantly impacts translation in bacteria, with IF-3 playing a crucial role in stress adaptation. In B. japonicum, which experiences variable conditions in soil and during nodule formation, IF-3 variants may differentially affect translation efficiency under stress. Researchers should investigate how temperature, pH, oxidative stress, and nutrient limitation affect IF-3 activity using ribosome profiling to identify stress-specific translational responses. The two-domain structure of IF-3, with its sequential binding mechanism , may undergo conformational changes under stress conditions, potentially altering ribosome binding kinetics. Comparative studies of IF-3 from different B. japonicum strains with varying stress tolerance (such as those adapted to different soil conditions) could reveal adaptations in translation initiation machinery. Researchers should also examine potential post-translational modifications of IF-3 during stress response, as these might fine-tune translation initiation to favor stress-response proteins while downregulating non-essential protein synthesis.

How does IF-3 contribute to translational regulation of symbiosis-specific genes in B. japonicum?

IF-3's role in translational regulation of symbiosis-specific genes in B. japonicum represents an important research frontier. Symbiosis islands (SI) in B. japonicum contain numerous genes essential for nodulation and nitrogen fixation, including nodD2D1ABCSUIJZ, noeEIL, nolAIKNOY, fixABCKRWX, and nifABDEHKNQSTWXZ . Research should focus on whether IF-3 exhibits differential affinity for mRNAs from these symbiosis genes compared to housekeeping genes. Ribosome profiling experiments comparing free-living B. japonicum to bacteroids in nodules could reveal IF-3-dependent translational regulation patterns. The potential role of IF-3 in recognizing specific features in the 5' untranslated regions of symbiosis genes would be particularly valuable to investigate. Since symbiosis requires precise timing of gene expression, researchers should examine whether IF-3 activity is modified during the transition from free-living to symbiotic states. Understanding this regulatory mechanism could provide insights for engineering improved nitrogen fixation efficiency in agricultural settings.

What are the most common pitfalls in studying recombinant B. japonicum IF-3 and how can they be addressed?

Studies involving recombinant B. japonicum IF-3 face several common pitfalls. Protein aggregation frequently occurs due to improper folding of the two-domain structure. To address this, researchers should optimize expression conditions (temperature, induction time, media composition) and consider fusion partners like MBP that enhance solubility. Proteolytic degradation at the flexible linker between N and C domains can be minimized by including protease inhibitors and reducing expression time. RNA contamination often affects purified IF-3 due to its RNA-binding properties; treatment with nucleases or high-salt washes can improve preparation purity. Loss of activity during purification may occur if the sequential binding mechanism is disrupted; researchers should verify activity through 30S binding assays before proceeding to more complex experiments . Finally, the high GC content of B. japonicum (63.34-63.55%) can cause codon usage issues in heterologous expression systems, which can be addressed through codon optimization or use of strains with rare tRNA supplements.

What controls are essential when studying the impact of IF-3 mutations on translation?

When studying the impact of IF-3 mutations on translation, several essential controls must be included. Wild-type IF-3 should always be tested in parallel with mutants under identical conditions to establish baseline activity. Empty vector controls are necessary to distinguish between effects of mutant IF-3 and background translational activity. Domain-specific mutations should be compared with linker mutations to differentiate the roles of the N and C domains in the sequential binding mechanism . Researchers should include controls for protein expression levels, as variations can confound interpretation of functional differences. Ribosome integrity controls are crucial when using purified components, as damaged ribosomes may show altered IF-3 interactions regardless of mutations. Temperature sensitivity controls help distinguish between mutations affecting protein stability versus those specifically impacting function. Finally, translation of different mRNA substrates (varying in Shine-Dalgarno sequences and start codon context) should be tested to comprehensively assess the impact of mutations on translation initiation fidelity.

How can researchers accurately quantify the binding kinetics of IF-3 to ribosomes from B. japonicum?

Accurately quantifying the binding kinetics of IF-3 to ribosomes from B. japonicum requires sophisticated biophysical approaches. Surface plasmon resonance (SPR) with immobilized ribosomes or ribosomal subunits allows real-time measurement of association and dissociation rates under varying conditions. For fluorescence-based approaches, researchers should carefully select labeling positions that don't interfere with the sequential binding mechanism of the C domain (G700 region) followed by the N domain (A790 region) . Microscale thermophoresis offers an alternative that requires smaller sample amounts and can measure interactions in solution. Time-resolved chemical probing, as demonstrated in previous research , provides detailed information about the sequence of binding events. Regardless of the method chosen, researchers must ensure ribosome preparations are homogeneous and functionally active. Controls should include competition with unlabeled IF-3 to verify specificity and measurements at different temperatures to determine thermodynamic parameters. Data analysis should employ models that account for the two-domain nature of IF-3 and potential conformational changes during binding.

What genomic approaches might reveal evolutionary adaptations in IF-3 across diverse Bradyrhizobium strains?

Future research into evolutionary adaptations of IF-3 across Bradyrhizobium strains should leverage comparative genomics approaches. Whole-genome sequencing of diverse strains from different geographic regions and host plants would enable comprehensive analysis of infC gene variations. Researchers should analyze selection pressure patterns across the infC coding sequence to identify regions under purifying versus diversifying selection. Genomic context analysis might reveal co-evolution of infC with other translation-related genes or host-specific factors. Phylogenomic approaches can place infC evolution in the context of Bradyrhizobium speciation events, such as the divergence of B. japonicum and B. diazoefficiens . Synteny analysis across the genomes of closely related species can provide insights into structural rearrangements affecting infC regulation. Transcriptomic analyses under various conditions would complement genomic data by revealing differential expression patterns of infC across strains. These approaches would help understand how translation initiation factor evolution contributes to the remarkable adaptability of Bradyrhizobium to diverse ecological niches and host plants.

How might systems biology approaches integrate IF-3 function into broader models of B. japonicum symbiotic adaptation?

Systems biology approaches offer powerful frameworks for integrating IF-3 function into holistic models of B. japonicum symbiotic adaptation. Multi-omics integration combining transcriptomics, proteomics, and metabolomics data from various stages of symbiosis establishment could reveal how IF-3-mediated translational control coordinates with transcriptional regulation. Network modeling approaches could identify regulatory hubs linking translation initiation to nodulation and nitrogen fixation gene clusters located on symbiosis islands . Agent-based modeling of B. japonicum-plant interactions could incorporate IF-3 activity parameters to simulate how translational regulation affects symbiotic efficiency under varying environmental conditions. Flux balance analysis extended to include translational efficiency constraints would help predict how IF-3 variants might affect metabolic adaptations during the transition from free-living to symbiotic states. These integrative approaches would provide a systems-level understanding of how fundamental cellular processes like translation initiation contribute to the complex phenomenon of symbiotic nitrogen fixation, potentially informing strategies to enhance agricultural productivity through improved bacterial inoculants.

What novel technologies might advance our understanding of IF-3 dynamics in living B. japonicum cells during symbiosis?

Emerging technologies offer exciting possibilities for studying IF-3 dynamics in living B. japonicum cells during symbiosis. Advanced microscopy techniques like super-resolution microscopy combined with split fluorescent protein tags could visualize IF-3 interactions with ribosomes in bacteroids within nodules. Microfluidic devices mimicking the rhizosphere environment would enable real-time observation of translational responses during host recognition and infection. In vivo RNA structure probing techniques could reveal how IF-3 affects mRNA structural rearrangements during translation initiation of symbiosis-specific transcripts. Optogenetic control of IF-3 activity would allow precise temporal manipulation of translation initiation during symbiotic development. Single-cell RNA-seq of bacteroids could correlate IF-3 expression levels with translation efficiency of specific symbiotic genes. CRISPR interference systems adapted for use in nodules could enable conditional knockdown of infC to study its role at specific symbiotic stages. These technological advances would provide unprecedented insights into how this fundamental translation factor contributes to the establishment and maintenance of the mutually beneficial relationship between B. japonicum and legume hosts.